Hyaluronan is a constituent of the extracellular matrix of connective tissue, and is actively synthesized during wound healing and tissue repair to provide a framework for ingrowth of blood vessels and fibroblasts. Changes in the serum concentration of hyaluronan are associated with inflammatory and degenerative arthropathies such as rheumatoid arthritis. In addition, hyaluronan has been implicated as an important substrate for migration of adhesion of leukocytes during inflammation.
Hyaluronan (hyaluronic acid, HA) is a high molecular mass polysaccharide that has ubiquitous distribution in the extracellular matrix, with highest concentrations in soft connective tissue. It is a linear polysaccharide comprising alternating glucuronic acid and N-acetylglucosamine residues linked by xcex2-1-3 and xcex2-1-4 glycosidic bonds (Laurent, T. C. et al. (1986), xe2x80x9cThe properties and turnover of hyaluronan.xe2x80x9d Functions of proteoglycans (Symposium, C. F., Ed. 124, Chichester, England). By interacting with other matrix molecules, such as chondroitin sulfate proteoglycans, hyaluronan provides stability and elasticity to the extracellular matrix. Hyaluronan has several physiochemical and biological functions such as space filling, lubrication, and providing a hydrated matrix through which cells can migrate (Toole, B. P. et al., Hyaluronate-cell interactions. The role of the extracellular matrix in development, (Trelstad, R. L., Ed., Alan R. Liss, New York (1984); Laurent, T. C. et al., Faseb J. 6:2397-2404 (1992)). Interaction of hyaluronan with the leukocyte cell surface receptor CD44 has been shown to contribute to organ specific leukocyte homing and migration (Jalkanen, S. T. et al., J. Cell. Biol., 105:893-990 (1987); Aruffo, A., et al., Cell 61:1303-1313 (1990); Culty, M. et al., J. Cell. Biol., 111:2765-2774 (1990); Miyake, K. et al., J. Exp. Med. 172:69-75 (1990); Sherman, L. et al., Current opinions in Cell Biology, 6:726-733 (1994)). Hyaluronan synthesis has been suggested to be required for cellular proliferation (Brecht, M. et al., Biochem. J. 239:445-450 (1986); Hronowski, L. and Anastassiades, T. P., J. Biol. Chem. 255:9210-9217 (1980); Matuoka, K. et al., J. Cell Biol. 104:1105-1115 (1987); Mian, N., Biochem. J. 237:333-342 (1986); Tomida, M. et al., J. Cell Physiol. 86:121-130 (1975)), and over-expression of receptors for hyaluronan, including a receptor for hyaluronan mediated motility (RHAMM) and CD44, correlates with increased levels of tumor metastasis (Gunthert, U., Curr. Topics Microbiol. Immunol. 184:47-63 (1993); Hall, C. L. et al., Cell 82:19-28 (1995); Turley, E. A., Cancer and Metastasis Reviews 11:1233-1241 (1992)). Purified preparations of hyaluronan exhibit unique viscoelastic properties, and as a consequence of these characteristics have been used in viscoelastic surgery and viscosupplementation (Balazs, E. A., and Denninger, J. L., Clinical uses of hyaluronan, The biology of hyaluronan, Ciba foundation symposium, Wiley, Chichester, England (1989)). Hyaluronan is synthesized mainly by mesenchymal cells and the accumulation of HA is an early event in tissue repair. The serum level of hyaluronan is elevated in inflammatory settings such as rheumatoid arthritis, osteoarthritis, liver cirrhosis, Werner""s syndrome, renal failure and psoriasis (Laurent, T. C. et al., Faseb J. 6:2397-2404 (1992); Laurent, T. C. Annals of Medicine 28:in press (1996)).
Hyaluronan is synthesized by a membrane bound synthase; monosaccharide and disaccharide residues are added to the reducing end of the polysaccharide as it protrudes through the plasma membrane (Prehm, P., Biochem. J. 211:181-189 (1983); Prehm, P., Biochem. J. 220:597-600 (1984)). Regulation of hyaluronan biosynthesis has been studied in several tissue culture systems. Factors involved in tissue growth and repair such as different isoforms of platelet derived growth factor (PDGF-AA, PDGF-BB), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor xcex2(TGF-xcex2), all exhibit stimulatory activity on hyaluronan biosynthesis (Heldin, P. et al., Biochem. J. 258, 919-922 (1992)).
A cDNA encoding a bacterial hyaluronan synthase has been cloned from Streptococcus pyogenes (hasA) (DeAngelis, J. P. et al., J. Biol. Chem. 268, 19181-19184 (1993)). Other related genes with N-acetylglucosaminyl transferase activity have been isolated from the nitrogen fixing bacteria Rhizobium (nodC) and chitin synthases (Chs) from Saccharomyces (DeAngelis, P. L. et al., Biochem. Biophys. Res. Comm. 199:1-10 (1994)). A putative vertebrate homolog, (DG42), was cloned from Xenopus laevis and has also been speculated to be a glycosaminoglycan synthetase (Rosa, F. et al., Develop. Biol. 129:114-123 (1988)). To date, however, a mammalian hyaluronan synthase gene has not been identified.
The present invention relates to isolated and/or recombinant nucleic acids which encode a mammalian hyaluronan synthase (e.g., human). In one embodiment, the nucleic acid of the present invention comprises SEQ ID NO:1. In another embodiment, the invention relates to a nucleic acid wherein said nucleic acid hybridizes under stringent conditions with a second nucleic acid having a nucleotide sequence of SEQ ID NO: 1.
The present invention also relates to a host cell comprising a nucleic acid encoding mammalian hyaluronan synthase. In a particular embodiment, the host cell comprises nucleic acid encoding mammalian hyaluronan synthase which is operably linked to an expression control sequence, whereby mammalian hyaluronan synthase is expressed when the host cell is maintained under conditions suitable for expression.
The present invention also relates to a method for producing a mammalian hyaluronan synthase comprising introducing into a host cell a nucleic acid construct comprising a nucleic acid which encodes a mammalian hyaluronan synthase, whereby a recombinant host cell is produced having said coding sequence operably linked to an (i.e., at least one) expression control sequence; and maintaining the host cells produced in a suitable medium under conditions whereby the nucleic acid is expressed.
The present invention also relates to an antibody or functional portion thereof (e.g., an antigen binding portion such as an Fv, Fab, Fabxe2x80x2, or F(abxe2x80x2)2 fragment) which binds mammalian hyaluronan synthase.
The present invention also relates to a method of detecting mammalian hyaluronan synthase in a sample comprising contacting a sample with an antibody which binds hyaluronan synthase under conditions suitable for specific binding of said antibody to the mammalian hyaluronan synthase; and detecting antibody-mammalian hyaluronan synthase.
The invention further relates to a method of using hyaluronan synthase to make hyaluronan.